Mosquito Control Strategies and Insecticide Resistance of the Malaria Vector in Urbanized Land Use Types in Suame Municipality, Ghana

Modified landscapes could create breeding habitats for mosquitoes and affect their community structure and susceptibility with implications for their management. Hence, in this study, household mosquito control methods in two urbanized landscapes; industrial and residential human settlements, in Ghana and insecticide susceptibility of the inhabiting Anopheles populations were assessed. Household knowledge and usage pattern of mosquito control methods in the modified landscapes were obtained using a questionnaire. Female adult Anopheles mosquitoes were also subjected to susceptibility tests using mosquito coils (0.08% meperfluthrin, 0.03% dimefluthrin, and 0.3% transfluthrin) and World Health Organization (WHO) insecticide test papers (0.05% deltamethrin, 4% DDT, 0.1% bendiocarb, 0.25% pirimiphos-methyl, and 5% malathion). Although insecticide-treated nets and aerosols were used for mosquito control, mosquito coils were the most common and widely preferred household method. The Anopheles mosquitoes were resistant to all the insecticides (mosquito coils and WHO test papers) except pirimiphos-methyl. Land use type did not affect insecticide resistance, but the insecticide type did. The findings indicate the effect of household insecticide usage practices on local mosquito populations and their implications for effective vector management and disease control in modified landscapes.


Introduction
Population increase leads to the creation of space for human settlement and industrialization, resulting in landscape modifcation.Tis alters the environment and may result in the establishment of habitats that promote the proliferation of mosquitoes [1,2].Hence, mosquitoes in contrasting land use types may respond diferently to control measures including insecticides due to the adaptation to contaminants in their breeding habitats [3][4][5][6][7] which may be occasioned by the modifed landscapes.Tis could impact on the choice of mosquito control method as well as the usage pattern, effectiveness [8] and eventually disease prevention and control.
Despite global improvements in vector control, mosquito-borne diseases still pose economic, health, and developmental challenges in many countries.Among these, malaria is one of the most fatal, causing about 608,000 deaths globally from an estimated 249 million cases in the year 2022 [9].Malaria, which is vectored by Anopheles mosquitoes, is an endemic and recurrent disease in Ghana with the entire population at risk.Controlling the vectors is an important strategy in curbing the disease.To achieve this, various techniques including insecticide use, avoidance of mosquitoinfested habitats, use of barriers like nets or screens, and environmental modifcation are employed [10,11].
In Ghana, the routinely used control methods in households include insecticide-treated (mosquito) nets (ITNs), mosquito coils, creams, and aerosols [12,13].However, mosquito coils are among the most preferred, preventing the entry of mosquitoes into houses as they create barriers which hinder host-seeking activity of the vectors [14], impair their ability to land and feed on hosts [15], and kill them.Te usage of mosquito coils is infuenced by several factors including perceived efectiveness, acceptance, and cost [13].Studies, however, indicate that these coils are inefective in combating mosquitoes as a sole control strategy and may have some adverse efects on humans [8,13,16,17].
Although several factors [13,18] drive household usage of domestic insecticides, the frequency of use may be due to the need to avoid mosquito nuisance since the sole use of some of these products is inefective in preventing mosquito bites [8].Te acceptance and efcacy of a mosquitocide may afect its usage pattern, and this could in turn impact on the environment, humans, and the target mosquitoes.Te usage pattern may however depend on the land use type due to the inherent vector burden of the modifed landscape.Mosquito diversity, behaviour, and response to insecticides could vary in diferent environments [4,8,[19][20][21] such as the diferential mortality levels observed in Anopheles gambiae populations from residential and industrial areas in Nigeria [22].Pyrethroid resistance in Anopheles mosquitoes in some residential urban areas in Ghana was also related to the high insecticide residues in their breeding habitats [4].Another study [8] in urban sites in southern Ghana also associated Culex insecticide resistance with household mosquito control strategies.It is therefore important to assess household practices regarding mosquito control and the efcacy of the most commonly used methods to inform vector management in diferent land use types.Te exposure to household insecticides and the associated pesticide challenge in the modifed landscapes may also contribute to resistance to nonhousehold insecticides such as active ingredients recommended by the World Health Organization (WHO) for indoor residual spraying (IRS).Hence, this study evaluated the household mosquito control methods in different land use types in Ghana and the insecticide susceptibility of the inhabiting Anopheles mosquitoes.

Study Area.
Te study was conducted in 2 diferent urbanized land use types in the Suame Municipality [21] of the Ashanti region of Ghana.Suame Magazine (06 °43′30″ N 1 °38′30″ W) is an auto-mechanic industrialized area with a residential and a nonresidential population of about 49,095 and 200,000, respectively, while Bremang (06 °43′60″ N 1 °38′60″ W) is a residential area with a population of 91,005 [23].Te study area is situated in the semideciduous forest zone and has two rainy periods (March to July and September to November) with a temperature range of 21.5 to 30.7 °C and relative humidity of 83.2%.

Knowledge, Attitude, and Perception of Insecticide Usage of Inhabitants.
A total of 171 and 326 questionnaires were administered in the industrial and residential area, respectively.Te industrial area was demarcated into 23 zones and 17 were selected for the sampling because the other zones had no residents.Houses within the 17 zones were numbered and the questionnaires (171) were distributed among the zones.In each zone, questionnaires were randomly administered based on the numbered houses.A similar process was conducted in the residential area.However, in Bremang, the 326 questionnaires were distributed among 3 suburbs within the locality (thus Bremang Fie, Bremang Universal Gospel Center, and Bremang Nkontwima) which are well laid out with numbered houses.Te questionnaire was used to obtain information on the demography, socioeconomics, knowledge, perception, control methods, and usage pattern of mosquito control methods of the households.

Mosquito Sampling and
Processing.Mosquito larvae were sampled from diverse breeding habitats (excavator bucket, streams, gutters, tyre casing, and metal containers) in the study area [21] from June to October 2019.Tese were sorted into their respective genera [21] and Anopheles larvae were transferred into disposable cups (6.2 × 6.0 cm) containing 100 ml of water.Te cups were placed in 40 cm 3   cages (1 cup per cage) and the larvae were fed with Raanan ® fsh pellet daily before pupation.Afterwards, female Anopheles that emerged was used for the bioassay tests.Te rearing temperature and humidity were between 25 and 30 °C and 75-90%, respectively.

Mosquito Coil Bioassay.
Efcacy of the commonly used mosquito coils (MC2: 0.03% dimefuthrin; MC6: 0.3% transfuthrin; and MC7: 0.08% meperfuthrin) in the households based on the questionnaire data was assessed in an exposure chamber (4 × 4 × 2.12 m; length × breadth × height).Twenty sugar-fed (10% sucrose solution on cotton wool) female [24] Anopheles mosquitoes (2-5 days old) were aspirated into 4 cages (30 × 30 × 30 cm).Each cage was hanged in the corner of the chamber at 20 cm from the ceiling and approximately 10 cm from the wall.Te mosquitoes were held for an hour before the coil was lit.A fan (Nasco ™ , China, diameter of head � 45.7 cm) at its minimum wind speed was placed in the middle of the chamber to circulate smoke emitted by the coil.Facing upwards, the fan was placed on the 2 Journal of Tropical Medicine foor and a plate with the coil was placed beside it.Te mosquito coil was lit to allow smoke emission before the fan was switched on.Te doors and windows of the exposure chamber were closed during the experiment.Te number of mosquitoes that were knocked down within 60 min was recorded.Afterwards, the mosquitoes were transferred from the cages into the holding tubes and fed ad libitum with cotton wool soaked in 10% sugar solution.Te mosquitoes were held at 27 ± 2 °C and 80 ± 10% relative humidity for 24 h and the observed mortality was recorded.Te test was replicated three times per mosquito coil.Te control experiment was conducted per this procedure; however, no coil was used [24].

WHO Insecticide Susceptibility Test.
Insecticide susceptibility of the female mosquitoes was determined using the WHO [25] standard tube assay.Five insecticideimpregnated papers from four insecticide classes approved by the WHO for IRS were used for the test.Tese were 0.05% deltamethrin (pyrethroid), 4% DDT (organochlorine), 0.1% bendiocarb (carbamate), 0.25% pirimiphosmethyl (organophosphate), and 5% malathion (organophosphate).Nonblood (10% sugar solution soaked on cotton wool) fed female Anopheles mosquitoes aged 2-5 days were exposed to each insecticide-impregnated paper and a control.Mosquitoes were exposed to the insecticides for 60 min and within that period, knockdown was recorded at 5 min intervals for 20 min and then at 10 min intervals for the rest of the 40 minutes.Mortality was recorded after 24 hours.Te test was replicated 4 times with 20 mosquitoes each.

Data Analysis.
Questionnaire data was inputted into Excel, exported to SPSS ver.23 and analyzed.Te % knockdown (%KD) was computed as the percentage of the number of knocked down mosquitoes compared to the total number of exposed mosquitoes per insecticide.Two-way analysis of variance (ANOVA) was used to compare the efect of land use type and mosquito coil brand on mosquito mortality.Te interaction efect of the two factors on mosquito mortality was also tested.Tukey HSD post hoc test was used to separate the means.ANOVA was run at a signifcance level of 5%.

Knowledge and Usage of Household Mosquito Control
Strategies.Many of the respondents in both land use types were females (Table 1).Majority (82.4%: industrial; 83.1% residential) of the respondents had basic and senior high school education and many of the respondents (91.8%: industrial; 96%: residential) were employed.Mosquito coil and repellent cream had the highest and lowest usage, respectively, in the study area (Table 2).Other mosquito control items used by respondents included mosquito spray/aerosol and insecticide-treated net (ITN) while a small number of the respondents (1.8%: industrial; 1.2%: residential) did not use any control method.Some unconventional methods such as burning herbs and the use of an electric fan were also employed (Table 2).In the industrial area, all respondents who use multiple mosquito control methods combined mosquito coil with other methods while 53.8% (43/80) of multiple control method users in the residential area use mosquito coil in combination with other methods (Tables 2 and 3).
Users of mosquito coils preferred specifc brands (Table 3).Te brands were coded in this study using the prefx MC (mosquito coil) and a number to avoid disclosing their identities.A few respondents (2.1%) in the industrial area used any brand/type that was available.Many respondents at the residential area preferred MC7 to the other brands.

Pattern of Mosquito Coil Usage and Perception of Side
Efects.Majority (65.5% industrial; 70.9% residential) of the coil users used at least one coil daily (Table 4).Te coils were usually used only indoors or both indoors and outdoors with many (80% industrial; 70.3% residential) of the respondents sleeping in a surrounding with a lit coil.Many (64.8% industrial; 51.7% residential) respondents reported that coils repel mosquitoes rather than kill them.At the industrial area, 65.5% reported that mosquito coils had some efects on humans while 34.6% of the respondents were not sure of the side efects of coils on humans (Table 5).In the residential area, about 79% of the respondents stated that coils had adverse efects on humans (Table 5) with some of the efects being cold, catarrh, cough, skin/eye irritation and breathing difculty.While cold and cough were the joint highest reported (17.9% each) in the industrial area, catarrh was the highest reported (33.7%) in the residential area.Skin/eye irritation was the least reported (2.1%) in the industrial area while choking (0%) was not reported in the residential area.

Perception of Mosquito Coil Efectiveness at the Auto-Mechanic Industrial and Residential
Areas.Many respondents (77.2%: industrial; 73.8%: residential) perceived mosquito coils to be efective and used them frequently (68.3%: industrial; 60.9%: residential) due to their efectiveness compared to their afordability, which was about 2fold lower (Table 6).

Mosquito Coil Efcacy.
In both land use types, Anopheles knockdown after 60 min was highest with MC6 and lowest with MC2 (Table 7).None of the mosquitoes in the control was knocked down after 60 min.After 24 h, mosquito mortality in the control was less than 5% (2.50 ± 1.25%: industrial; 3.33 ± 1.10%: residential).Tere was a 30-67% mosquito mortality when exposed to the mosquito coils (Table 7).Tere was no signifcant infuence of land use type on Anopheles mortality (F 1,16 � 0.279, p � 0.603), but the type of mosquito coil signifcantly afected Anopheles mortality (F 3,16 � 581, p � 0.001).Te interaction efect of land use type and mosquito coil on mosquito mortality was signifcant (F 3,16 � 2.85, p � 0.007).Te performance of each mosquito coil was similar within the two land use types, except MC6 which recorded a higher and signifcantly diferent mortality at the residential area compared to the auto-mechanic area (p < 0.05).
Journal of Tropical Medicine 3.5.WHO Insecticide Susceptibility.Among the 5 insecticide papers, pirimiphos-methyl had the highest % knockdown after 5 min of exposure (Figures 1 and 2) and this trend continued to the 60 th min (42.5%:industrial; 43.75%: residential).DDT recorded the lowest % knockdown (8.75%: industrial; 12.5%: residential) on the mosquitoes.For all the insecticides, the % knockdown increased rapidly from the 15 th to the 60 th min.

Discussion
Although households in the study area use diferent mosquito control methods, mosquito coil usage was the most prevalent in both land use types, an observation reported in communities in the Ashanti, Bono East, Central, Greater Accra, Upper East, and Western regions in Ghana [8,12,13,17].Tis can be attributed to factors such as its perceived efectiveness, availability, and afordability [13] compared to the other control methods.Te median cost of    spatial repellent to protect one person per year is similar to that of ITN and lower than that of IRS [11].Usage of a combination of mosquito control methods was high, and this usually revolved around the use of mosquito coils with the other methods.Tis demonstrates the integral role of mosquito coils in household mosquito control and the inability of a single household method to confer the desired antimosquito protection, hence augmenting with other methods to increase the level of protection [13,16,26].
Mosquito coil usage was mostly on a daily basis in both land use types.Tis is similar to fndings in other parts of Ghana [12,13,17].Te daily usage is suggestive of the persistent threat posed by mosquitoes in the study area and the constant need for protection.Although most coil users usually lit one coil, the proportion of people who used more than one coil was higher in the industrial area.Tis probably indicates a higher mosquito threat in the industrial area since our previous study [21] observed a higher mosquito abundance in this land use type.While most of the residential households used coils indoors, those in the industrial area used them in both indoor and outdoor environments.It is likely that the mosquitoes in the residential area are largely   6 Journal of Tropical Medicine endophagous and endophilous while in the industrial area, there is a sympatry of both indoor and outdoor feeding and resting mosquitoes.Most coil users sleep in the presence of a burning coil.Tis practice is common in several countries [26][27][28] and exposes users to coil emissions.Although Hogarh et al. [26] estimated a low environmental health risk associated with the usage of some coils, coil users in our study reported adverse efects on their skin, eyes, and respiratory function.Such efects have also been reported in other studies [12,13,17,28,29].Mosquito coils are advertised as inducers of mosquito death; however, most users regarded them as efective in repelling rather than killing mosquitoes [12,13,17].Users generally regarded the coils as efective in mosquito avoidance and the efectiveness based on this attribute was the most predominant reason for its usage.
Among the mosquito coils used, 0.3% transfuthrin induced a higher knockdown and mortality than the others.Tis is similar to a study by Bibbs et al. [30] where transfuthrin and meperfuthrin were observed to have the highest vapour toxicity on some mosquito species compared to other volatile pyrethroids.Although majority of the respondents cited efectiveness of coils in controlling mosquitoes as the reason for their usage, our results show that the Anopheles mosquitoes were resistant to the insecticide coils.Tis result is consistent with previous reports of Anopheles resistance to mosquito coils [12,13,16,26].Hence, this relates to the users' perception of repellency and not mortality as the most predominating coil property.
Although the land use type did not infuence mosquito mortality, the coil type did, with 0.3% transfuthrin inducing the highest mortality.Mosquito coil brands used the industrial area were more than the residential area.Tis could be due to the experimentation of diferent coil types by households in the quest for the most efective in the industrial area.Diferential mortality efect on the mosquitoes may be due to the type of active ingredient and resistance to the insecticide coils could be attributed to the excessive and indiscriminate use of domestic insecticides in the study area.Exposure to heavy metal pollutants can increase insecticide resistance in mosquitoes [5]; hence, metal pollutants and other contaminants in water of mosquito breeding sites within the industrial area could have partly contributed to the lower mortality.
Te WHO insecticide test results indicated that for each insecticide, mosquito mortality from the residential area was higher than the industrial area although this was not signifcant between the two sites.A similar pattern in mosquito mortality was observed between Anopheles gambiae populations from industrial and residential areas in Kano, Nigeria for malathion and bendiocarb [22].Also, except pirimiphos-methyl, mosquitoes from the two land use types in our study were resistant to the insecticides.Mortality of the mosquitoes to the insecticides was signifcantly diferent in each land use type, although the mortality response to bendiocarb was not signifcantly diferent from those to deltamethrin and malathion.Resistance of Anopheles mosquitoes to insecticides has been reported around the country over the past decades [31][32][33][34], and these insecticides continue to be inefective in controlling mosquitoes as observed in our study.Most of these previous studies were conducted in agricultural areas, thereby attributing mosquito resistance to chemical contamination during agricultural activities.Hunt et al. [32] detected Anopheles resistance/suspected resistance to 4 classes of insecticides (pyrethroids, carbamates, organophosphates, and organochlorines) in four mining areas in Ghana although susceptibility was detected to one organophosphate active ingredient.Similarly, our study shows resistance of Anopheles mosquitoes to active ingredients in the four insecticide classes (i.e., <90% mortality) except the organophosphate pirimiphos-methyl.Resistance to bendiocarb and DDT have also been observed in modifed landscapes such as industrial and residential sites in Nigeria [22] and diferent agricultural production sites in Cote D'Ivoire [35].However, the Anopheles mosquitoes in the study in Nigeria were susceptible to malathion [22] unlike the populations in our study and that of two vegetable cultivation sites in Kouadio et al. [35].
Te questionnaire responses showed frequent use of ITNs, aerosols, and mosquito coils in the study area.Tis practice might have contributed to pyrethroid resistance [4,8] due to the selection pressure.Continuous exposure of mosquitoes to pyrethroid insecticides can induce metabolic enzymes [36,37], potentially resulting in the development of resistance.Insecticide contamination of mosquito breeding habitat has also been implicated in mosquito resistance in an urban residential area with no agricultural activity [4].Although, the current study did not focus on associating resistance with breeding site contamination, it is important to explore this phenomenon to gain more understanding in this regard.Te susceptibility of Anopheles mosquitoes to the organophosphate insecticide (pirimiphos-methyl) in our study substantiates previous fndings [32][33][34].Tus, given the resistance of the mosquitoes to pyrethroids, the organophosphate pirimiphos-methyl can be used as an alternative in Indoor Residual Spraying.

Conclusion
Mosquito coil forms an important component of household antimosquito measures with diverse brands used at the automechanic industrial area compared to the residential area.Even though coils were regarded as efective, mosquito repellency was the most widely reported efect compared to mosquito mortality.Anopheles mortality was not signifcantly afected by land use type despite the lower mortality at the industrial area.However, mortality was signifcantly afected by insecticide type.Te mosquitoes were resistant to all the insecticides (including the mosquito coils) except pirimiphos-methyl.Te current study contributes valuable information on Anopheles resistance towards insecticides for vector management, highlighting the potential impact of household insecticide use on resistance development and the implications for community-wide vector control using indoor residual spraying.
Approval.Ethical clearance was obtained from the Committee on Human Research, Publication and Ethics (Ref: CHRPE/AP/334/19) of the Kwame Nkrumah University of Science and Technology and the Suame Municipal Assembly (Ref: SMA/GA.1/14/01/10).

Table 2 :
Household mosquito control methods at the study area.
N: number of respondents.

Table 3 :
Mosquito coil brands used at the industrial and residential areas.
N: number of respondents.

Table 1 :
Demographic characteristics of respondents at the study area.

Table 5 :
Side efect perception of mosquito coil usage at the auto-mechanic industrial and residential areas.

Table 6 :
Efectiveness of mosquito coils and factors that infuence usage.
*No response from 3 respondents from the residential area.

Table 7 :
Susceptibility of Anopheles mosquitoes to pyrethroid-based mosquito coils.
KD: knockdown; SE: standard error.Values with diferent lowercase superscripts in the same column are signifcantly diferent (p < 0.05).Values with diferent uppercase superscripts are signifcantly diferent (p < 0.05) across columns and rows.Post hoc test was done using Tukey HSD.

Table 4 :
Frequency and pattern of mosquito coil usage at the auto-mechanic industrial and residential areas.

Table 8 :
[25]ality of mosquitoes 24 h after 1 h exposure to insecticides.: standard error.Values with diferent superscripts in the same column are signifcantly diferent (p < 0.05) as determined by Tukey HSD post hoc test.RS: resistance status.R: resistant.S: susceptible.*Basedon the WHO susceptibility criteria[25]. SE